The goal of this research proposal is to examine the role of transcriptional silencers of the osteocalcin gene in osteoblast like cell lines. The system selected to pursue these studies is the rat osteocalcin gene in osteoblast like cell lines. The system selected to pursue these studies is the rat osteosarcoma cell line ROS17/2.8, which has properties of mature osteoblasts and expresses the endogenous osteocalcin gene. These studies will focus on the TTTCTTT silencing motif, which is located at +118 to +124 in the first intron activity in the osteoblast like cell lines ROS 17/2.8 and UMR 106. The transient expression of a series of osteocalcin- luciferase fusion genes with site directed mutations in the TTTCTTT silencer motif into ROS 17/2.8 cells will define the residues critical for the silencing activity of this motif, as outlined in the first specific aim. Additional, gel retardation assays and Southwestern blot analysis using nuclear extracts from RO2 17/2.8 cells, as described in specific aim 2, will determine the DNA-protein interactions with the TTTCTTT motif that lead to its silencing activity. Based on the results of these studies, strategies for the cloning or purification of the protein interacting with this motif will be devised.